iav h1n1 strain a pr8 8 34  (ATCC)

Journal: iScience

Article Title: Ambient temperature regulates CD4 + T cell tonic T cell receptor signaling and responsiveness

doi: 10.1016/j.isci.2026.115801

Figure Lengend Snippet: Thermoneutrality-driven exacerbation of inflammatory disease severity is linked with increased CD4 + T cell TNF production (A–D) 8-to-10-week-old WT C57BL/6J male mice were fed a high-fat diet (HFD) and housed in Tn- or Ts-conditions for 20 weeks (n = 9–22 biological replicates per group). (A) Schematic overview. (B-D) The following parameters were quantified at the time of terminal harvest: Total body weight (B); serum alanine transaminase (ALT) levels via calorimetric assay (C); and frequency of TNF + hepatic CD4 + T cells (TCRβ + TCRγδ − NK1.1 − CD4 + ) upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA; 50 ng/mL) and ionomycin (1 mg/mL) via flow cytometry (D). (E–H) 8-to-10-week-old WT C57BL/6J male mice were housed in Tn- or Ts-conditions. After 2 weeks, mice were intranasally infected with influenza virus (Charles River Influenza A/PR/8/34 [H1N1], Batch: 4XP160913; 30 Hemagglutinin units/mice; n = 5–7 biological replicates per group). (E) Schematic overview. (F) Body weight change post-infection. (G, H) The following parameters were quantified 5 days post-infection: bronchioalveolar lavage fluid (BALF) IL-6 levels via ELISA (G); and frequency of TNF + pulmonary CD4 + T cells (CD45 + TCRβ + TCRγδ − NK1.1 − CD8 − ) upon ex vivo stimulation with PMA (50 ng/mL) and ionomycin (1 mg/mL) via flow cytometry (H). (I-L) 12-week-old WT C57BL/6J male mice were housed in Tn- or Ts-conditions. After 2 weeks, mice were intratracheally treated with house dust mite (HDM; 10 μg/mouse) on days 0, 14, and 21 (n = 8–11 biological replicates per group). (I) Schematic overview. (J–L) The following parameters were quantified 3 days after final HDM exposure: BALF eosinophil count via morphological analysis (J); IL-5 production of single lung cell suspensions after 72-h ex vivo cell culture (K); and frequency of TNF + pulmonary CD4 + T cells (CD45 + TCRβ + TCRγδ − NK1.1 − CD8 − ) upon ex vivo stimulation with PMA (50 ng/mL) and ionomycin (1 mg/mL) via flow cytometry (L). (B-D, F-H, J-L) Mann-Whitney U test. ∗: p < 0.05, ∗∗∗: p < 0.001, and ∗∗∗∗: p < 0.0001. For bar and line graphs, data represent mean ± SEM.

Article Snippet: Mice were sedated with 2.5% isoflurane (Akorn, NDC 59399-106-01) and treated intranasally with 30 HA Units/7.16 log 10 EID/5.7 log 10 TCID50 H1N1 PR8 influenza (Charles River; Influenza A/PR/8/34, Batch: 4XP160913; provided by Dr. William Zacharias, CCHMC) resuspended in 0.9% sodium chloride solution (Hospira, NDC 0409-4888-02).

Techniques: Ex Vivo, Flow Cytometry, Infection, Virus, Enzyme-linked Immunosorbent Assay, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: S1PR1 signaling biases neutrophils toward long-lived low-inflammatory functional states

doi: 10.64898/2026.02.05.703783

Figure Lengend Snippet: (A) Schematic of high-dose H1N1 (LD 50 ) model over 21 days. (B) Survival curve of LysM -S1pr1 TG (n = 20) and control (n = 18) mice in response to H1N1 infection. Survival curves were compared using the Gehan–Breslow–Wilcoxon test. (C) Body weight loss of LysM -S1pr1 TG (n = 8) and control (n = 8) mice. (D) Blood oxygen saturation (SpO 2 ) in LysM -S1pr1 TG (n = 9) and control (n = 11) mice post-infection. Both (C) and (D) were tested by two-way mixed-effects analysis with Geisser–Greenhouse correction and Šídák’s multiple comparisons test; * p < 0.05, ** p < 0.01. (E) Schematic of the low-dose (0.16 LD 50 ) H1N1 model at 3 and 7 dpi. (F) Total cell count in BALF at 3 and 7 dpi (n = 7 mice per group). (G and H) BALF neutrophil and macrophage count at 3 and 7 dpi (n = 7 mice per group). (I-K) Expression of viral genes, including nucleoprotein (NP), polymerase acidic protein (PA), and hemagglutinin (HA), in the infected lung in control (n = 3) and LysM -S1pr1 TG (n = 4) mice. (L) Total protein in BALF in control (n = 3) and LysM -S1pr1 TG (n = 6) mice at 7 dpi. (M) IL-6 (ng/mL) secretion in BALF in control (n ≥ 4) and LysM- S1pr1 TG (n ≥ 5) mice at 3 and 7 dpi. (N) IL-10 (ng/mL) secretion in BALF in control (n ≥ 4) and LysM -S1pr1 TG (n ≥ 3) mice at 3 and 7 dpi. Data are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t test).

Article Snippet: Influenza A/Puerto Rico 8/1934 (PR8) H1N1 strain was obtained from Charles River (Cat# 10100374, Wilmington, MA), aliquoted, and stored in liquid nitrogen.

Techniques: Control, Infection, Cell Characterization, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: S1PR1 signaling biases neutrophils toward long-lived low-inflammatory functional states

doi: 10.64898/2026.02.05.703783

Figure Lengend Snippet: (A) Kaplan–Meier survival curves of Mrp8- S1pr1 transgenic (TG) mice ( n = 25) and Cre-negative littermate controls ( n = 21) following intranasal infection with high-dose H1N1 (LD 50 ). Survival was monitored for up to 21 days post-infection. Statistical significance was assessed using the log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests. (B) Body weight change in control (n = 13) and Mrp8- S1pr1 TG (n = 21) mice following influenza infection, expressed as a percentage of the initial body weight. Data represent mean ± SD. (C) Arterial oxygen saturation (SpO₂) measured longitudinally after infection using pulse oximetry in control (n = 13) and Mrp8- S1pr1 TG (n = 21) mice. Data represent mean ± SD. Statistical significance is denoted as * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Influenza A/Puerto Rico 8/1934 (PR8) H1N1 strain was obtained from Charles River (Cat# 10100374, Wilmington, MA), aliquoted, and stored in liquid nitrogen.

Techniques: Transgenic Assay, Infection, Control

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: Pathogenesis dynamics and transcriptomic changes during influenza infection in mice. ( A ) Left: the PCA for the sequencing samples at 5 dpi with various infection dose. Right: the PCA for the sequencing samples with 100 or 1,000 PFU PR8 IAVs infection during the time post-infection (1, 3, 5, and 7 dpi). ( B ) Volcano plots of the DEGs between PBS and IAVs treated groups (10 1 , 10 2 , 10 3 , and 10 4 PFU, respectively). The abscissa represents the log 2 FC, the ordinate represents the −log 10 ( P adj). Red points are the upregulated DEGs defined by the absolute value of FC > 2 and P adj < 0.05, while blue points are the downregulated DEGs. ( C and D ) Heatmap clustering of key extracellular-matrix-related genes from overlap DEGs across various infection doses at 5 dpi ( C ) and across multiple timepoints in the 100 PFU infection group ( D ). Expression levels are shown as log 2 -transformed FPKM values after centralization correction, highlighting the top 30 genes with significant changes relative to the PBS group. ( E ) WGCNA module identification and trait correlation, positive and negative correlations are indicated by red and blue colors, respectively. ( F ) Gene interaction network within the ME-darkred module, where node color depth indicates the number of connections. The central area highlights candidate hub genes.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Sequencing, Expressing, Transformation Assay

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: CTSS is upregulated by influenza infection in mouse lung. ( A ) CTSS mRNA expression in lung tissues from mice ( n = 3 per group) inoculated with either PBS or 100 PFU of PR8 virus, quantified by RT-qPCR. ( B ) Western blotting analysis of proteins in lung tissues from mice inoculated with PBS or 100 PFU of PR8 virus. ( C ) CTSS activity in BALF and lung homogenates from mice inoculated with PBS or 100 PFU of PR8, measured using a CTSS Activity Assay Kit. ( D ) Left panel: comparison of CTSS mRNA expression levels in the lungs of mice infected with 5 PFU and 100 PFU of IAV, determined by RT-qPCR. Right panel: western blotting analysis of activated CTSS protein levels in lung tissues from mice infected with 5 PFU and 100 PFU of PR8. ( E ) Temporal profile of CTSS mRNA expression across various days post-infection, measured via RT-qPCR (left), and western blotting analysis of activated CTSS protein levels across different timepoints post-infection (right). Data are represented as mean ± standard deviation (SD). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Expressing, Virus, Quantitative RT-PCR, Western Blot, Activity Assay, Comparison, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: The effects of CTSS inhibitor LY3000328 on influenza infection in mice. ( A ) Schematic diagram of the experimental design. C57BL/6J mice ( n = 5 per group) received intraperitoneal injection of 30 mg/kg LY3000328 or a placebo vehicle twice daily for 5 days. Four hours after the initial dose, mice were intranasally inoculated with 100 PFU of PR8 virus. A mock control group received the vehicle orally twice daily and PBS intranasally. Body weight was monitored daily. Mouse lungs were collected at 5 dpi for the analyses of viral titers, histopathology, and gene expression. ( B ) CTSS activity in lung homogenate among different groups. ( C ) Viral titers in lung homogenates determined using the TCID 50 assay in MDCK cells. ( D ) Body weight change of mice across the experimental period. ( E ) Survival curve of mice across the experimental period. ( F ) Histopathological examination of mouse lungs performed by H&E staining. ( G ) Analysis of mRNA expression levels of cytokines in lung tissues by RT-qPCR. Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, non-significant.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Injection, Virus, Control, Histopathology, Gene Expression, Activity Assay, Staining, Expressing, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: Influenza infection activates CTSS expression in lung epithelial cells (A549). ( A ) CTSS mRNA expression in A549 cells following infection with PR8 virus at MOIs of 0, 0.1, 0.5, and 1. ( B ) Protein levels of pro-CTSS and activated CTSS in A549 cells 24 hpi with serial MOIs of PR8 virus, assessed by western blotting. ( C ) CTSS activity in the supernatant and cell lysate of A549 cells infected with indicated MOI of PR8 virus at 24 hpi, measured using a CTSS Activity Assay Kit. Data are presented as mean ± SD from three independent experiments. ***, P < 0.001; ****, P < 0.0001; ns, non-significant.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Expressing, Virus, Western Blot, Activity Assay

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: CTSS does not affect PR8 virus replication but reduces cytokine expression in A549 cells. A549 cells were transfected with two different CTSS-siRNAs (siCTSS #1 and siCTSS #2) or a scramble siRNA (siSCR) and then infected with 0.25 MOI of PR8 virus. ( A ) Left panel: representative western Blot analysis of CTSS and viral proteins (HA and NP) expression at 24 hpi, Right panel: intensity scanning of the bands from the left, quantified with data from three independent repeats. ( B ) Viral M gene expression at 24 hpi was quantified by RT-qPCR. ( C ) Viral titers at specified timepoints were measured using the TCID 50 assay. ( D ) The changes in mRNA expression levels of CTSS, TNF-α, RANTES, and IP-10 at 24 hpi were assessed by RT-qPCR. Data are presented as mean ± SD. Two to three independent experimental repeats were conducted for each assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Virus, Expressing, Transfection, Infection, Western Blot, Gene Expression, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: CTSS is released into the cytoplasm during influenza infection, thereby regulating apoptosis and epithelial barrier integrity. ( A ) Cell death was assessed by measuring LDH release from non-targeting and CTSS-targeting siRNA transfected A549 cells following PR8 infection at 24 hp i, using an LDH Cytotoxicity Assay Kit (Roche, USA, 91963) according to the manufacturer’s instructions. ( B ) TUNEL staining and quantification of positive cells in non-targeting and CTSS-targeting siRNA-transfected A549 cells after PR8 virus infection at 24 hpi. Representative images (left panel) and statistical data (right panel) are shown. ( C ) Protein levels of CTSS, LAMP1, NP, and HA and PARP1 in the cytoplasm, lysosome, and supernatant of A549 cells infected with PR8 (MOI = 0.1) at 0, 6, 12, and 24 hpi were determined by western blotting. ( D ) CTSS activity in the cytoplasm, lysosome, and supernatant of A549 cells infected with PR8 virus was measured using a CTSS Activity Assay Kit. ( E ) Expression of CTSS, intercellular tight junctions, and cell death markers in non-targeting and CTSS-targeting siRNA transfected A549 cells following PR8 virus infection at 24 hpi was determined by western blotting. Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Transfection, LDH Cytotoxicity Assay, TUNEL Assay, Staining, Virus, Western Blot, Activity Assay, Expressing